BACKGROUND: Simultaneous determination of different natures of analytes is of great significance for saving sample volumes and simplifying analytical procedures. However, sample preparation for the simultaneous extraction of polar and non-polar analytes represents a challenge in sample preparation. Inspired by the successive liquid-phase microextraction (sLPME) method for acidic and basic analytes that we previously developed, we first proposed an efficient successive electromembrane extraction (sEME) system by adjusting the acidity of the donor solution and using binary organic solvents for extraction of polar and non-polar targets from biological samples in this work. RESULTS: We performed a detailed optimization of the sEME system. Here, carnitine (C0) and acylcarnitines were selected as model analytes since the demand increased especially in metabolomics studies. The combination of 2-nonanone and 2-nitrophenylpentyl ether (NPPE) was selected as supported liquid membranes (SLMs), and trichloroacetic acid (TCA) 100 % (v/v) was added to donor solution to adjust the acidity of the donor solution after the first sEME process (sEME-1). The recoveries of the targets in blood and urine were 47%-119% and 54%-118%, respectively. Moreover, the sEME systems were evaluated by liquid chromatography tandem mass spectrometry (LC-MS/MS) from biological samples. The limit of detection (LOD) and limit of quantitation (LOQ) of analytes were 0.03-1.33 ng mL SIGNIFICANCE: sEME enabled the extraction of polar and non-polar analytes from the same sample under optimal extraction conditions for all target analytes, which provided ideas for efficient sEME of exogenous and endogenous analytes from biological samples for forensic, clinical, and epidemiological studies.